酿酒酵母利用甘蔗糖蜜异源合成L-乳酸的关键问题解析

Molasses is a main byproduct during sugarcane making process, and more than 200 million tons of molasses each grinding season is produced in Guangxi province, where contributes to the major production of sugar in China. To date, limited research work has been conducted on the resource utilization of molasses. L-lactic acid is widely used in food, medicine, textile, leather, cosmetics and chemical industries, and has a prosperous economic value. To date, L-lactic acid is mainly fermented by lactic acid bacteria or chemical synthesized, however, the two manners all have scabrous problems that restrained efficiency production of this compound. In the present study, we propose to produce L-lactic acid in an engineered Saccharomyces cerevisiae strain which utilize molasses as the main carbon resource. Production of L-lactic acid could be firstly achieved by expression of heterologous gene that encoding lactate dehydrogenase (LDH) in S. cerevisiae. On this basis, genetic manipulations would be conducted to enhance the carbon flux to the biosynthesis of L-lactic acid in S. cerevisiae, such as gene knocking out, regulating NADH/NAD+ ratio and screening of proper transporters which could efficient transport L-lactic acid outside organelle, and so on. Finally, key factors that restrain the fermentation process from molasses would also be analyzed and solved, to increase L-lactic acid production in S. cerevisiae. The results obtained from this study would be helpful to the production of L-lactic acid in S. cerevisiae, and would also be useful to increase the added value of molasses.

糖蜜是白糖炼制过程中的主要副产物，广西是全国耕地白糖的主产区，糖蜜年产量在200万吨以上，当前关于甘蔗糖蜜的资源化利用的研究较为缺乏。L-乳酸广泛应用于食品、医药、纺织、皮革、化妆品及化工领域，具有较高的经济价值。目前，L-乳酸主要由乳酸菌发酵生产或经化学合成获得，但都存在其不可解决的难题。本项目拟通过代谢工程改造酿酒酵母利用甘蔗糖蜜生产L-乳酸进行系统研究，通过表达乳酸脱氢酶编码基因（LDH），在酿酒酵母体内构建一条L-乳酸合成途径。在此基础上，利用基因敲除手段，调控代谢流更多的流向L-乳酸合成途径。通过调控酿酒酵母体内的辅因子NADH/NAD+的比例、筛选适合的L-乳酸转运蛋白等手段，逐步提高L-乳酸的产量。最后，分析并解决甘蔗糖蜜对酿酒酵母代谢生产L-乳酸的限制因素，提高L-乳酸得率。本研究所得的相关成果将丰富酿酒酵母代谢工程改造生产L-乳酸的机制，提高甘蔗糖的附加产值。

L-乳酸在食品、医药等领域具有广泛应用前景。本研究以酿酒酵母（Saccharomyces cerevisiae）CEN.PK2-1B为研究对象，异源表达源于牛的乳酸脱氢酶(LDH)。重组菌株利用甘蔗糖蜜实现了L-乳酸的积累。通过对丙酮酸代谢旁路途径的修饰以及相关辅因子的调节，考察了副产物的积累情况以及菌体生长状况。主要研究成果如下：.（1）将源于牛并经密码子优化后的LDH在S. cerevisiae菌株以及缺失PDC1的S. cerevisiae菌株内表达并在预处理过的甘蔗糖蜜培养基中培养。重组菌发酵36 h的L-乳酸产量分别为2.01 g/L及2.38 g/L。实验结果表明，表达外源LDH的酿酒酵母可在利用甘蔗糖蜜实现L-乳酸的生产；.（2）为了降低L-乳酸前体丙酮酸的旁路代谢。本研究构建失活了PDC1的菌株以及同时缺失PDC1和ADH1的菌株。结果发现，前者的乙醇产量为9.58 g/L，后者的乙醇产量为8.83 g/L，转化率分别为0.227 g/g，0.201 g/g，相比于出发菌株分别降低29.94%、37.96%，改造后菌株的生物量相比于出发菌株基本不变。实验结果表明，敲除PDC1与ADH1能够降低副产物乙醇的转化率，且不影响菌株的生长；.（3）由于PDC1、ADH1的缺失会导致S. cerevisiae胞质acetyl-CoA产量降低以及乙醛的过量积累。通过过表达乙醛脱氢酶6（Acetaldehyde dehydrogenase 6，ALD6）编码基因及异源表达源于大肠杆菌的丙酮酸氧化酶（Pyruvate oxidase，poxB）编码基因，修饰丙酮酸代谢旁路途径，把乙醛引向acetyl-CoA合成方向，降低菌体负荷。结果发现，ALD6的过表达使乙醇转化率降低12.94%，菌体生物量提高31.12%；异源表达源于大肠杆菌的poxB虽然没能改变改造菌株菌体生物量，但能使乙醇转化率降低5.97%；.（4）酿酒酵母中，乙醇合成能力的下降会引起胞质NADH/NAD+值升高，导致甘油过量积累。通过超表达aMDH3，提高S. cerevisiae胞质苹果酸脱氢酶（Malate dehydrogenase，MDH）酶活，提高了苹果酸-天冬氨酸脱氢酶穿梭途径速率。实验结果发现，甘油的产量降低17.56%，乙醇转化率降低25.71%。